Major Activities: This project uses small animal models, which allow us to undertake targeted questions, as well as studies of healthy human cells and studies of patients who have sustained lymphocyte depletion. This bench-to-bedside-to-bench approach provides insights that increase basic understanding and set the stage for clinical application. Specific Objectives: Specific objectives of this project are to: 1) improve understanding of the biology of T cell depletion and 2) develope new therapies to improve immune reconstitution or that could replicate the beneficial aspects of lymphopenia while avoiding long-term detrimental effects. Significant Results: We have previously demonstrated that age-, disease- and therapy-associated effects on thymic function fundamentally limit T cell immune reconstitution in humans following lymphocyte depletion. We also previously demonstrated that thymic-independent pathways of immune restoration can largely restore immune competence and are well poised for manipulation in the context of immunotherapy for cancer. We previously discovered that interleukin-7 is the major driver of the thymic-independent pathway and have therefore focused efforts on clinical development of recombinant human IL-7. We have also tested the hypothesis that immune reconstitution can be modulated favorably in tumor bearing hosts by adoptive transfer of large numbers of resting T cells that have been manipulated to contain diminished numbers of suppressive cells. The first major accomplishment of this project during FY13 was publication of a landmark report that identified a novel role for soluble IL7 receptor in modulating bioactivity of IL7. This project, focused on studies of the biology of IL7 receptor, was driven by the genetic observation that polymorphisms IL7R effect susceptibility to multiple sclerosis, but there was no understanding of the biological basis for these findings. Through these studies, we found that genetic variations in IL7R that predispose to autoimmunity results in a higher splicing rate of IL7R, and increased copies of mRNA lacking the transmembrane domain (e.g. exon 6). We next predicted that polymorphisms predisposing to autoimmunity would be associated with higher levels of soluble IL7R. We tested this hypothesis using cohorts of healthy and MS-afflicted patients, and found that autoimmunity predisposing polymorphisms led to higher circulating levels of soluble IL7Ra. Furthermore, when soluble IL7Ra was added to cultures containing rhIL7, bioactivity of rhIL7 was diminished in the short-term but increased over the longer term (e.g. days), due to diminished IL7 consumption in the presence of soluble IL7Ra. We also observed qualitative differences in IL7 signaling in the presence of soluble IL7Ra, with diminished levels of SOCS and Fas induced by IL7 signaling in this context. Both SOCS and Fas are negative regulators of T cell function, consistent with a model where higher levels of soluble IL7Ra leads to enhanced T cell activity, through both prolongation of the IL7 signal and diminished negative regulators. Together, this led to the hypothesis that polymorphisms in IL7R increase the risk of autoimmunity by enhancing the bioactivity of IL-7. Using clinical samples from healthy donors and from patients with multiple sclerosis, we confirmed that soluble IL7R levels are tightly regulated by genetic polymorphisms in IL7Ra and that in patients with multiple sclerosis, IL-7 levels themselves are modulated by polymorphisms in IL7Ra. Finally, we observed in a mouse model of autoimmune encephalitis, that co-administration of sIL7Ra increased the potency of IL-7 induced disease, thus providing a clear basis for implicating soluble IL7Ra levels in susceptibility to multiple sclerosis. These studies provide novel, fundamental insights implicating an essential role for IL7R in modulating IL7 bioactivity in vivo, and are the first to explain how genetic polymorphisms in IL7Ra increase susceptibility to multiple sclerosis. They also lead to the prediction that co-treatment with IL7 plus sIL7Ra will enhance the potency of IL7 as a therapeutic agent. Finally, these data reveal the fundamental insight that healthy humans have a profound molar excess of soluble IL7Ra compared to IL7 itself. This is very different than the relationship between soluble receptor found for IL2 and IL15 and likely explain the prolonged half-life of IL7 when used as a therapeutic agent. The second major accomplishment of this project in FY13 was completion of the first two cohorts of a clinical trial wherein rhIL7 was administered to a large cohort of children with cancer after primary front line therapy. This represents the only clinical experience with rhIL7 in children thus far. We demonstrated the agent to be safe and highly effective at increasing the pace and degree of immune reconstitution following intensive chemotherapy for cancer. We also conducted state-of-the-art studies using next generation sequencing to enumerate the T cell receptor repertoire of rhIL7 vs non-IL7 treated patients. These studies demonstrate diversification of the repertoire with recombinant human interleukin-7, confirming what was previously postulated using approaches of much lower resolution. Furthermore, as discussed in Project III, exciting clinical results suggest that this approach may improve survival in this high-risk population.